Crystal form A of 2-butoxy-7-(4-(pyrrolidin-1-ylmethyl)benzyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine for inhibiting toll-like receptor activity

ABSTRACT

The present invention relates to crystalline form A of a TLR7 agonist 2-butoxy-7-(4-(pyrrolidin-1-ylmethyl)-benzyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine (formula I), a method for preparing the crystalline form A, and the use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. patent application Ser. No.16/075,297 filed on Aug. 3, 2018, which is a national phase ofInternational Application No. PCT/CN2017/072891, filed on Feb. 4, 2017,which claims priority to Chinese Patent Application No. 201610082029.8,filed on Feb. 5, 2016, each of which is incorporated herein by referencein its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

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INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON COMPACT DISC

Not Applicable

BACKGROUND OF THE INVENTION (1) Field of the Invention

The present invention relates to the field of medicinal chemistry, andparticularly relates to crystal form A of a TLR7 agonist(2-butoxy-7-(4-(pyrrolidin-1-ylmethyl)benzyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine),a preparing process thereof, a crystalline composition comprising thecrystal form A, a pharmaceutical composition comprising the crystal formA or the crystalline composition and use thereof.

(2) Description of Related Art

Toll-like receptor is expressed by various immune cells and recognizeshigh reserved structural motifs: Pathogen Associated Molecular Pattern(PAMP) expressed by microorganism pathogens or Damage AssociatedMolecular Patterns (DAMP) released by dead cells. PAMP or DAMPstimulates Toll-like receptor to trigger signal cascade which inducesthe activations of transcriptional factors like AP-1, NF-κB andinterferon regulators (pulse response function). It results in variouscell responses, including productions of interferons, proinflammatorycytokines and effector cytokines, whereby immune response is produced.By far, 13 types of Toll-like receptors have been discovered in mammal.Toll-like receptors 1, 2, 4, 5 and 6 are mainly expressed on the cellsurface while Toll-like receptors 3, 7, 8 and 9 are expressed in theendosome. Different Toll-like receptors recognize ligands derived fromdifferent pathogens. Toll-like receptor 7 (TLR7) is mainly expressed byplasmaeytoid dendritic cells (pDC), and recognized via ligand to inducethe secretion of interferon α (IFN-α). Toll-like receptor 7 (TLR7) andToll-like receptor 8 (TLR8) are highly homologous and therefore theligand of TLR7 in most cases is also that of TLR8. TLR8 stimulationmainly induces the productions of cytokines like tumor necrosis factor α(TNF-α) and chemoattractant. Interferon α is one of the medicines fortreating chronic hepatitis B or hepatitis C while TNF-α is aproinflammatory cytokine, of which the over secretion will result severeside effects.

There have been reported several TLR7 agonists, like imiquimod (BritishJournal of Dermatology 2003; 149 (Suppl. 66): 5-8), resiquimod(Antiviral Research 64 (2004) 79-83), GS-9620 (Gastroenterology (2013),144(7), 1508-1517). Nevertheless, it is desirable to have novel TLR7agonists with better selectivity, activity and safety.

Chinese Patent Application No. 201410405136.0 which is incorporated inits entirety by reference herein discloses one small molecule, i.e.2-butoxy-7-(4-(pyrrolidin-1-ylmethyl)benzyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine,which has the following structure:

BRIEF SUMMARY OF THE INVENTION

In an aspect, provided is crystal form A of the compound of formula I:

The crystal form A is characterized by an X-ray powder diffraction(XRPD) pattern having diffraction peaks at 2θ=5.5°±0.2°, 10.1°±0.2°,13.8°±0.2°, 19.7°±0.2°, 23.7°±0.2°, 24.1°±0.2°.

In another aspect, provided is a process for preparing crystal form A ofthe compound of formula I, comprising the following steps:

1) dissolving the compound of formula I in a crystallizing solvent,which is preferably heated to promote dissolution; and

2) cooling for crystallization, filtrating, washing and drying to obtainthe crystal form A.

In another aspect, provided is a pharmaceutical composition, comprisingthe crystal form A or the crystalline composition thereof according tothe invention. The pharmaceutical composition can further optionallycomprise pharmaceutically acceptable carrier, excipient and/or medium.

In another aspect, provided is a method for treating or preventingToll-like receptor 7 (TLR7) associated disease, comprising administeringto a subject in need thereof the crystal form A or the crystallinecomposition thereof or the pharmaceutical composition according to theinvention in an effective amount. Preferably, the disease is virusinfection.

In a further aspect, provided is use of the crystal form A or thecrystalline composition thereof or the pharmaceutical compositionaccording to the invention for the manufacture of a medicament fortreating or preventing Toll-like receptor 7 (TLR7) associated disease.Preferably, the disease is virus infection.

In a yet further aspect, provided is the crystal form A or thecrystalline composition thereof or the pharmaceutical compositionaccording to the invention for use in treating or preventing Toll-likereceptor 7 (TLR7) associated disease. Preferably, the disease is virusinfection.

In one embodiment of the invention, the virus infection is hepatitisvirus infection, particularly hepatitis B or hepatitis C virusinfection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: XRPD pattern of the crystal form A of the compound of formula I.

FIG. 2: Unit cell diagram of the crystal form A of the compound offormula I.

FIG. 3: DSC pattern of the crystal form A of the compound of formula I.

DETAILED DESCRIPTION OF THE INVENTION General Definition and Terminology

Unless stated otherwise, the terms and phrases used herein have thefollowing meaning. A specific term or phrase shall not be considered asunclear or indefinite when it is not specifically defined. It should beunderstood according to the general meaning. The trade name used hereinrefers to the corresponding product or the active ingredient.

Unless specifically defined otherwise, proportion (including percentage)or part is calculated based on weight herein.

When used with a numerical variable, the term “approximate” or “about”usually refers to the value of the variable and all the values of thevariable within the experimental error (for example, within an average95% confidence interval) or within ±10% of the specified value, or awider range.

The expression “comprise” or its synonyms “contain”, “include”, “have”or the like is open-ended, which does not exclude other unlistedelements, steps or ingredients. The expression “consist of” excludes anyunlisted elements, steps or ingredients. The expression “substantiallyconsist of” refers to specified elements, steps or ingredients within agiven range, together with optional elements, steps or components whichdo not substantively affect the basic and novel feature of the claimedsubject matter. It should be understood that the expression “comprise”encompasses the expressions “substantially consist of” and “consist of”.

The term “optional” or “optionally” means the event described subsequentthereto may or may not happen. This term encompasses the cases that theevent may or may not happen.

The term “pharmaceutical composition” refers to an active ingredient,which is optionally combined with one or more pharmaceuticallyacceptable components (for example, but not limited to carrier and/orexcipient). The active ingredient is exemplified as the compound offormula I or salt thereof, the crystal form according to the invention,or the crystalline composition according to the invention.

The term “pharmaceutically acceptable carrier” refers to those carrierswhich have no significant irritation and do not impair the bioactivityand property of the active compound. The “pharmaceutically acceptablecarrier” refers to inert substance which is administered with activeingredient and is beneficial to the administration thereof, andcomprises but not limited to any of the following substances approved byState Food and Drug Administration for use in human or animal (e.g.livestock): glidant, sweetening agent, diluent, preservative,dye/colorant, flavoring agent, surfactant, wetting agent, dispersant,disintegrant, suspending agent, stabilizing agent, isotonic agent,solvent or emulsifying agent. Non-limiting examples of the carrierscomprise calcium carbonate, calcium phosphate, various sugars andstarches, cellulose derivative, gelatine, vegetable oil and polyethyleneglycol or the like. Other information regarding the carriers may befound in Remington: The Science and Practice of Pharmacy, 21st Ed.,Lippincott, Williams & Wilkins (2005), of which the contents areincorporated herein by reference. The term “excipient” generally refersto the carrier, diluent and/or medium used to formulate effectivepharmaceutical composition.

The term “administration” or “administrating” or the like refers to amethod that enables a compound or composition to be delivered to adesired site of biological action. Such methods comprise but not limitedto oral, parenteral (including intravenous, subcutaneous,intraperitoneal, intramuscular, intravascular injection or infusion),local, rectal administration or the like.

As for pharmaceutical or pharmacological active agent, the term“effective amount” refers to the amount of the medicament or agent whichis not toxic but sufficient to achieve the desired effect. With respectto the oral formulation herein, the “effective amount” for an activesubstance in the composition refers to the amount required to achievethe desired effect in combination with another active substance in thecomposition. The effective amount may be determined individually anddepends on the age and general condition of the receptor as well asspecific active substance. The effective amount in specific case can bedetermined by a person skilled in the art through conventional test.

The term “active ingredient”, “therapeutic agent”, “active substance” or“active agent” refers to a chemical entity useful for treating orpreventing target disorder, disease or condition effectively. The termherein may refer to for example the compound of formula I or saltthereof, the crystal form according to the invention or crystallinecomposition according to the invention.

In X-ray powder diffraction (XRPD or XRD) spectra, the diffractionpattern obtained from crystalline compound is generally characteristicfor a particular crystal form in which the relative intensities of thebands (especially at low angles) may vary with the dominant orientationeffect due to the difference of crystallization conditions, particlediameters, and other measuring conditions. Therefore, the relativeintensities of diffraction peaks are not characteristic for the givencrystal form. It is more important to note the relative positions ofpeaks rather than their relative intensities when determining whetherthe crystal form is the same as that known in the art. In addition,there may be slight errors in the positions of the peaks for any givencrystal form, which is also well known in the art of crystallography.For example, the position of the peak may shift due to the change intemperature, sample movement or instrument calibration during analysisof the sample; and the measuring error of 2θ value may sometimes beabout ±0.2°, typically about ±0.1°. Therefore, this error should betaken into account when determining the crystal structure. If thecrystal forms according to the invention are described as substantiallyas shown in the figure, the term “substantially” is also intended toencompass such differences in the diffraction peak.

In the XRPD pattern, the peak position is usually represented by angle2θ or crystal surface distance d and a simple conversion between d and θis d=λ/2 sin θ, where d represents the crystal surface distance, λrepresents the wavelength of the incident X-ray, and θ is diffractionangle. As for the same crystal form of the same compound, the peakpositions of the XRPD pattern are similar as a whole, and the relativeintensity error may be large. It should also be noted that, inidentification of a mixture, some diffraction lines may be lost due tothe factors like decrease in content, etc., and thus it is not necessaryto rely on the entire bands observed in the high purity sample, and evenone band may be characteristic for a given crystal.

In the present invention, X-ray powder diffraction pattern is determinedas follows: apparatus: Bruker D8 ADVANCE X-ray diffractometer; method:target: Cu: K-Alpha; wavelength λ=1.54179 Å; voltage: 40 kV; current: 40mA; scanning range: 4˜40°; sample rotation speed: 15 rpm; scanningspeed: 10°/min.

Differential scanning calorimetry (DSC) is used to measure thetransition temperature when a crystal absorbs or releases heat due tochanges in its crystal structure or crystal melting. The thermaltransition temperature and the melting point error are typically withinabout 5° C., usually about 3° C. for the same crystal form of the samecompound in a continuous analysis. When a compound is described ashaving a given DSC peak or melting point, it means the DSC peak ormelting point±5° C. Provided is an auxiliary method by DSC to identifydifferent crystal forms. Different crystal forms may be identifiedaccording to their different transition temperature characteristics. Itis noted that the DSC peak or melting point of the mixture may vary overa wide range. In addition, the melting temperature is associated withthe rate of temperature rise due to the decomposition during the meltingof the substance.

Differential scanning calorimetry (DSC) herein is measured by thefollowing method: apparatus: TA Q2000 differential scanning calorimeter;method: a sample (˜1 mg) is placed in a DSC aluminum pan, method: 25°C.˜300° C., heating rate 10° C./min.

The crystal form according to the invention can also be characterized bycell parameters. Such parameters can be determined by single crystalX-ray crystallographic analysis. For example, the detailed informationof cell parameters can be found in Chapter 3 of Stout & Jensen, X-Raystructure Determination: A Practical Guide, MacMillian Co., New York,N.Y. (1968).

The cell parameters herein are determined by the following method:Diffractometer: Rigaku MicroMax-007HF; Wavelength: 1.54178 Å;Temperature: 296K.

The term “crystalline composition” refers to a solid form, whichcomprise the crystal form A according to the invention. The amounts ofthe crystal form A contained in the crystalline composition may be 50%or more, 80% or more, 90% or more, or 95% or more. In addition tocrystal form according to the invention, the crystalline composition mayalso optionally comprise other crystal or amorphous form of the compoundof formula I or the salt thereof or the impurities other than thesesubstances. It should be understood by those skilled in the art that thesum of contents of the components in the crystalline composition shouldbe 100%.

Crystal Form A

Provided is crystal form A of the compound of formula I, which hasdiffraction peaks at 2θ=5.5°, 10.1°, 13.8°, 19.7°, 23.7°, 24.1°±0.2° inX-ray powder diffraction (XRPD) pattern.

In a specific embodiment, the crystal form A has diffraction peaks at2θ=5.5°, 10.1°, 13.8°, 16.4°, 19.7°, 23.7°, 24.1°, 27.9°±0.2° in X-raypowder diffraction (XRPD) pattern.

In a more specific embodiment, the crystal form A has diffraction peaksat 2θ=5.5°, 10.1°, 13.8°, 16.4°, 17.9°, 19.0°, 19.7°, 20.3°, 21.8°,22.1°, 23.7°, 24.1°, 25.5°, 27.9°, 32.9°, 34.0°±0.2° in X-ray powderdiffraction (XRPD) pattern.

In a particular embodiment, diffraction peaks of the crystal form A ofthe compound of formula I are characterized as follows:

2θ ± 0.2 Relative intensity Number (°) (%) 1 5.5 100.0 2 10.1 52.4 310.8 4.2 4 13.8 35.8 5 14.9 2.7 6 16.4 12.2 7 17.9 6.1 8 18.6 2.6 9 19.08.2 10 19.7 20.1 11 20.3 9.2 12 21.0 4.3 13 21.8 6.6 14 22.1 6.4 15 23.737.1 16 24.1 17.4 17 25.5 9.9 18 27.3 4.3 19 27.9 13.4 20 28.6 3.1 2132.9 5.1 22 34.0 5.3

In an embodiment, the X-ray powder diffraction pattern of the crystalform A of the compound of formula I is substantially shown in FIG. 1.

In another embodiment, the crystal form A of the compound of formula Ihas the following cell parameters (as shown in FIG. 2):

-   -   a=16.560 (3) Å    -   b=10.426 (2) Å    -   c=12.203 (2) Å    -   α=90°    -   β=98.54(3)°    -   γ=90°    -   Space groups: P2₁/c    -   Z=4.

The crystal form A can also be characterized by DSC, with initialtemperature of 199.0° C. and peak temperature of 200.4° C.

In an embodiment, DSC pattern of the crystal form A of the compound offormula I is shown in FIG. 3.

Preparation Process

Provided is also a process for preparing the crystal form A of thecompound of formula I, comprising precipitating the crystal form A ofthe compound of formula I from a solvent.

In an embodiment, the process comprises the following steps:

1) dissolving the compound of formula I in a crystallizing solvent,which is preferably heated to promote dissolution; and

2) cooling for crystallization, filtrating, washing and drying to obtainthe crystal form A.

In step 1), the crystallizing solvent is selected from the groupconsisting of methanol, ethanol, propanol, isopropanol, n-butanol,isobutanol, tertiary butanol, acetone, ethyl acetate, water and mixedsolvent thereof; preferably ethanol.

In step 1), the amount of crystallizing solvent added per 1 g of thecompound of formula I is 2 to 10 mL, preferably 4 to 8 mL, morepreferably 5 to 7 mL.

In step 1), heating is used to form a homogeneous system of the compoundof formula I and the crystallizing solvent. The heating temperature maybe 40° C. to 90° C., preferably 50° C. to 80° C., more preferably 70° C.to 80° C.

Provided is also a crystalline composition, comprising the crystal formA of the compound of formula I. In an embodiment, based on the weight ofthe crystalline composition, the content of crystal form A is 50% ormore, preferably 80% or more, more preferably 90% or more, and mostpreferably 95% or more. The crystalline composition, in addition to thecrystal form A, can also comprise the compound of formula I or saltthereof in other crystal or amorphous forms, or impurities other thanthese substances.

Pharmaceutical Composition and Administration

Provided is a pharmaceutical composition, which comprises the crystalform A of the compound of formula I or the crystalline compositionthereof in an effective amount. Furthermore, the pharmaceuticalcomposition also may or may not comprise pharmaceutically acceptablecarrier, excipient and/or medium.

The compound according to the invention is administrated in a pure formor in a suitable pharmaceutical composition form, which can be performedvia any acceptable administration mode of the agent with similar use.Pharmaceutical composition according to the invention may be prepared bycombining of the compound according to the invention or the salt thereofwith a suitable pharmaceutically acceptable carrier, for example it maybe formulated into solid, semi-solid, liquid or gas formulation, such astablet, pill, capsule, powder, granule, ointment, emulsion, suspension,solution, suppository, injection, inhalant, gel, microsphere, aerosol orthe like.

The pharmaceutical composition according to the invention may beprepared by the processes well-known in the art, such as conventionalmixing, dissolution, granulation, dragee coating, levigation, emulsion,freeze-drying or the like.

Typical routes for administering the compound according to the inventionor the pharmaceutical composition thereof comprise but not limited tooral, rectal, transmucosal, enteral administration or local,transcutaneous, inhalant, parenteral, sublingual, intravaginal,intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous,intravenous administration.

In a preferred embodiment, the pharmaceutical composition is in an oraladministration form. As for oral administration, the active compoundsmay be mixed with the pharmaceutically acceptable carriers, excipientsand/or media well-known in the art to prepare the pharmaceuticalcomposition. The carriers, excipients and media may be used to preparethe compounds according to the invention into tablet, pill, troche,dragee, capsule, liquid, gel, slurry, suspension or the like useful fororal administration to the patient.

Solid oral composition may be prepared by conventional mixing, fillingor compressing processes, for example, by the following processes:mixing the active compound with solid excipients, optionally milling theresultant mixture, adding other proper adjuvants if necessary, and thenprocessing the mixture into granules so as to obtain the core of tabletor dragee. The proper excipients comprise but not limited to filler,such as sugar, including lactose, sucrose, mannitol or sorbitol;cellulose preparation such as microcrystalline cellulose, maize starch,wheat starch, rice starch and potato starch; and other substances, suchas silica gel, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, carboxymethyl cellulose sodium and/or polyvinylpyrrolidone;disintegrant, such as carboxymethyl starch sodium, croscarmellosesodium, crosslinked polyvinylpyrrolidone, agar or alginic acid. Saltsuch as sodium alginate may also be used. The core of the dragee may beoptionally coated through well-known processes in general pharmaceuticalpractice, especially by enteric coating.

Beneficial Effects

The crystal form A of the compound of formula I according to theinvention has the advantages of high purity, high crystallinity and goodstability. Meanwhile, the preparation process of the crystal form Aaccording to the invention is simple, the solvent is cheap and easilyobtainable, the crystal form condition is mild, and the process issuitable for industrial production.

The technical solutions of the invention are illustrated according tothe following paragraphs [1] to [20]:

[1] A crystal form A of the compound of formula I,

characterized in that it has diffraction peaks at 2θ=5.5°±0.2°,10.1°±0.2°, 13.8°±0.2°, 19.7°±0.2°, 23.7°±0.2°, 24.1°±0.2° in X-raypowder diffraction pattern.

[2] The crystal form A of the compound of formula I according toparagraph [1], characterized in that it has diffraction peaks at2θ=5.5°±0.2°, 10.1°±0.2°, 13.8°±0.2°, 16.4°±0.2°, 19.7°±0.2°,23.7°±0.2°, 24.1°±0.2°, 27.9°±0.2° in X-ray powder diffraction pattern.

[3] The crystal form A of the compound of formula I according toparagraph [2], characterized in that it has diffraction peaks at2θ=5.5°±0.2°, 10.1°±0.2°, 13.8°±0.2°, 16.4°±0.2°, 17.9°±0.2°,19.0°±0.2°, 19.7°±0.2°, 20.3°±0.2°, 21.8°±0.2°, 22.1°±0.2°, 23.7°±0.2°,24.1°±0.2°, 25.5°±0.2°, 27.9°±0.2°, 32.9°±0.2°, 34.0°±0.2° in X-raypowder diffraction pattern.

[4] The crystal form A according to any one of paragraphs [1]-[3],characterized in that, it has X-ray powder diffraction patternsubstantially shown in FIG. 1.

[5] The crystal form A according to any one of paragraphs [1]-[4],characterized in that, it has the following cell parameters:

-   -   a=16.560 (3) Å    -   b=10.426 (2) Å    -   c=12.203 (2) Å    -   α=90°    -   β=98.54(3)°    -   γ=90°    -   Space groups: P2₁/c    -   Z=4.

[6] The crystal form A according to any one of paragraphs [1]-[5],characterized in that, when characterized by DSC, the initialtemperature is 199.0° C.±5° C. and the peak temperature is 200.4° C.±5°C.

[7] A process for preparing the crystal form A according to any one ofparagraphs [1]-[6], comprising the following steps:

1) dissolving the compound of formula I in a crystallizing solvent,which is preferably heated to promote dissolution; and

2) cooling for crystallization, filtrating, washing and drying to obtainthe crystal form A.

[8] The preparing process according to paragraph [7], characterized inthat the crystallizing solvent in step 1) is selected from the groupconsisting of methanol, ethanol, propanol, isopropanol, n-butanol,isobutanol, tertiary butanol, acetone, ethyl acetate, water and mixedsolvent thereof.

[9] The preparing process according to paragraph [8], characterized inthat the crystallizing solvent is ethanol.

[10] The preparing process according to any one of paragraphs [7]-[9],characterized in that, in step 1), the amount of crystallizing solventadded per 1 g of the compound of formula I is 2 to 10 mL.

[11] The preparing process according to paragraph [10], characterized inthat the amount of crystallizing solvent added per 1 g of the compoundof formula I is 4 to 8 mL.

[12] The preparing process according to paragraph [11], characterized inthat the amount of crystallizing solvent added per 1 g of the compoundof formula I is 5 to 7 mL.

[13] The preparing process according to any one of paragraphs [7]-[12],characterized in that, in step 1), heating is used to form a homogeneoussystem of the compound of formula I and the crystallizing solvent.

[14] The preparing process according to any one of paragraphs [7]-[13],characterized in that, in step 1), the heating temperature may be 40° C.to 90° C.

[15] The preparing process according to paragraph [14], characterized inthat, in step 1), the heating temperature may be 50° C. to 80° C.

[16] The preparing process according to paragraph [15], characterized inthat, in step 1), the heating temperature may be 70° C. to 80° C.

[17] A crystalline composition, characterized in that, based on theweight of the crystalline composition, the crystal form A of thecompound of formula I according to any one of paragraphs [1]-[6] is 50%or more, preferably 80% or more, more preferably 90% or more, and mostpreferably 95% or more.

[18] A pharmaceutical composition, comprising the crystal form Aaccording to any one of paragraphs [1]-[6] or the crystallinecomposition according to paragraph [17] in an effective amount.

[19] Use of the crystal form A of the compound of formula I according toany one of paragraphs [1]-[6] or the crystalline composition accordingto paragraph [17] or the pharmaceutical composition according toparagraph [18] for the manufacture of a medicament for treatingToll-like receptor 7 (TLR7) associated disease.

[20] The use according to paragraph [19], characterized in that thedisease is virus infection, particularly the virus infection ishepatitis virus infection, for example hepatitis B or hepatitis C virusinfection.

EXAMPLES

The following abbreviations are used herein: SEM-Cl:2-(trimethylsilyl)ethoxymethyl chloride; SEM:2-(trimethylsilyl)ethoxymethyl; DIPEA: diisopropylethylamine; TFA:trifluoroacetic acid; DMF: N,N-dimethylformamide; n-BuOH: n-butanol;NH₃.H₂O: aqueous ammonia; Na: sodium; XRPD: X-ray powder diffraction;DSC: differential thermal analysis.

The solvents used herein are commercially available and can be usedwithout further purification. The synthesis reactions in preparationexamples are generally performed under inert nitrogen atmosphere inanhydrous solvent. Data of proton magnetic resonance is recoded inBruker Avance III 400 (400 MHz) spectrometer, with the chemical shiftshown as (ppm) at tetramethylsilane low field. Mass spectrometry isdetermined on Agilent 1200 plus 6110 (&1956A). LC/MS or Shimadzu MSincludes a DAD: SPD-M20A(LC) and Shimadzu Micromass 2020 detector. Massspectrometer is equipped with an electrospray ionization (ESI) operatedat positive or negative mode.

Preparation Example 1 Preparation of2-butoxy-7-(4-(pyrrolidin-1-ylmethyl)benzyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amineFormula III 2,4-dichloro-5-((2-(trimethylsilyl)ethoxy)methyl)-5H-pyrrolo[3,2-d]pyrimidine

The compound of formula II (2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidine)(4.00 kg, 21.28 mol) was dissolved in DMF (20.00 L), and DIPEA (2.58 kg,20.00 mol) was added in portions at room temperature (25° C.) followedby stirring for 30 min. The reaction liquid was cooled to 0° C. with anice bath, and then SEM-Cl (4.00 kg, 24.00 mol) was added dropwise slowlyover 5 h at a dropping rate of 1 to 2 drops per second. After addition,the reaction liquid was stirred at 0° C. for 4 h. The reaction wasmonitored by HPLC. After completion, the reaction liquid was quenchedand diluted with 70 L of water and then extracted with ethyl acetate (15L×3). The combined organic phase was washed successively with 1 Maqueous hydrochloric acid (5 L×2) and saturated brine (7 L×2), and thesolvent was removed by distillation under reduced pressure to give thecompound of formula III (6.40 kg, 20.11 mol, yield 94.50%).

¹H NMR (400 MHz, DMSO-d₆) δ 8.24-8.35 (m, 1H), 6.70-6.85 (m, 1H), 5.77(s, 2H), 3.45-3.57 (m, 2H), 0.74-0.86 (m, 2H), 0.00 (s, 9H).

Formula IV2-chloro-5-((2-(trimethylsilyl)ethoxy)methyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine

The compound of formula III (1.60 kg, 5.03 mol) was dissolved inisopropanol (1.60 L) in a 10 L clave. Aqueous ammonia (4 L) was added atroom temperature (25° C.) in one portion and the reaction mixture wasstirred at 95° C. for 7 h. The reaction was monitored by HPLC. Aftercompletion, the reaction liquid was allowed to cool to room temperatureand filtered through a Buchner funnel to give a dark brown solid. Thesolid was successively slurried with ethyl acetate/n-heptane (1/1, 5L×2) and ethyl acetate (4 L) to give the compound of formula IV as brownsolid (1.25 kg, 4.18 mol, yield 83.1%).

¹H NMR (400 MHz, DMSO-d₆) δ 7.61-7.77 (m, 1H), 6.97-7.19 (m, 2H),6.28-6.38 (m, 1H), 5.54-5.67 (m, 2H), 3.43-3.53 (m, 2H), 0.76-0.91 (m,2H), 0.07 (s, 9H).

Formula V2-butoxy-5-((2-(trimethylsilyl)ethoxy)methyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine

To n-BuOH (17.0 L) was slowly added metal sodium (525.05 g, 22.84 mol)in portions under nitrogen. After addition, the temperature of thesystem was raised to 60° C., and stirring was performed continuously atthe temperature until the metal sodium was completely dissolved. Thenthe system was cooled to 25° C., and the compound of formula IV (1.95kg, 6.53 mol) was added in portions. After being mixed homogenously withstirring, the reaction mixture was continuously stirred for 8 h at 90°C. The reaction was monitored by HPLC. After completion, the reactionmixture was allowed to cool spontaneously to 25° C., and slowly pouredinto 30 L of saturated aqueous ammonium chloride. Then the reactionmixture was extracted with ethyl acetate (15 L×3) and the combinedorganic phase was washed with saturated brine (20 L×2), dried withanhydrous Na₂SO₄, and filtered. After the solvent was distilled offunder reduced pressure, the residue was slurried in n-heptane (4 L) andthe solid was separated by filtration and then slurried in ethyl acetate(5 L) to give the compound of formula V as yellow-white solid (1.53 kg,4.55 mol, 69.7%).

¹H NMR (400 MHz, DMSO-d₆) δ 7.49-7.54 (m, 1H), 6.54-6.62 (m, 2H),6.15-6.20 (m, 1H), 5.54 (s, 2H), 4.10-4.22 (m, 2H), 3.42-3.55 (m, 2H),1.58-1.73 (m, 2H), 1.35-1.47 (m, 2H), 0.90-0.96 (m, 3H), 0.83-0.89 (m,2H), 0.05 (s, 9H).

Formula VI: 2-butoxy-5H-pyrrolo[3,2-d]pyrimidin-4-amine

The compound of formula V (1.10 kg, 3.27 mol) was dissolved in TFA (5.50L) and the reaction liquid was stirred at 25° C. for 16 h. The reactionwas monitored by HPLC. After completion, TFA was removed by distillationunder reduced pressure and the residue was dissolved in methanol (1.2 L)and ice water (1.2 L). pH of the system was adjusted to 12 withconcentrated aqueous ammonia under uniform stirring. The mixture wasstirred for 2 h and the precipitate was precipitated from the solutioncontinuously. After filtration, the filter cake as white solid wasslurried with 15% aqueous ammonia (1.2 L×3) and ethyl acetate (4 L)successively to give the compound of formula VI as white solid (550.00g, 2.67 mol, 81.7%).

¹H NMR (400 MHz, methanol-d₄) δ 7.37 (d, J=2.89 Hz, 1H), 6.29 (d, J=3.01Hz, 1H), 4.27 (t, J=6.53 Hz, 2H), 1.75 (d, J=7.91 Hz, 2H), 1.44-1.61 (m,2H), 1.00 (t, J=7.40 Hz, 3H).

Formula VII4-((4-amino-2-butoxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-hydroxymethyl)benzaldehyde

To a three-necked flask were added terephthalaldehyde (790.64 mg, 5.82mmol) and isopropanol (10 mL),2-butoxy-5H-pyrrolo[3,2-d]pyrimidin-4-amine (1.00 g, 4.85 mmol) wasadded with stirring, and the system was cooled to 0° C. and stirred foranother 10 min. Purified water (10 mL) and potassium carbonate (804.17mg, 5.82 mmol) were added, and reacted at 25° C. for 16 h until the rawmaterials were depleted with the monitor by LCMS. Solid was precipitatedout after the reaction was completed. After filtration, the resultingsolid was slurried with 20 mL of purified water and 30 mL (ethylacetate/n-heptane=1/20) successively, filtered and dried to give thecompound of formula VII as yellow solid (1.50 g, 4.41 mmol, yield:90.9%).

¹H NMR (400 MHz, methanol-d₄) δ 9.94 (s, 1H), 7.86 (d, J=8.16 Hz, 2H),7.72 (d, J=8.16 Hz, 2H), 7.12-7.17 (m, 1H), 6.19 (s, 1H), 4.28 (t,J=6.53 Hz, 2H), 1.68-1.77 (m, 2H), 1.44-1.54 (m, 2H), 0.97 (t, J=7.34Hz, 3H).

Formula VIII(4-amino-2-butoxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)(4-(pyrrolidin-1-ylmethyl)phenyl)methanol

To a 30 L clave were added the compound of formula VII (450.0 g, 1.32mol) and isopropanol (4.5 L), and the mixture was stirred for 5 min.Then glacial acetic acid (119.0 g, 1.98 mol) was added and thetemperature of the system was lowered to 0-10° C. with stirring.Pyrrolidine (112.4 g, 1.58 mol) was added dropwise, with the temperaturebelow 10° C. After addition, sodium triacetoxyborohydride (420.0 g, 1.98mol) was added in portions and reacted at 10-20° C. for 3 h until theraw materials were depleted with the monitor by liquid chromatography.After the completion of the reaction, 5 L of purified water was addedand the temperature of the solution was lowered to about −10° C., and 12L of 15% aqueous ammonia was added to the solution, with the solutiontemperature below 0° C. during addition. Solid was precipitated outunder stirring. Filtration was performed and the resulting filter cakewas slurried with 2 L of water and 2 L×2 ethyl acetate. Filtration wasperformed and drying was conducted under reduced pressure at 40° C. for12 h to give the compound of formula VIII as yellow solid (465.0 g, 1.18mol, yield 89.4%, moisture 0.9%).

¹H NMR (400 MHz, methanol-d₄) δ 7.46 (d, J=7.91 Hz, 1H), 7.29 (d, J=8.03Hz, 1H), 7.09 (s, 1H), 6.12 (s, 1H), 4.29 (t, J=6.53 Hz, 2H), 3.60 (s,2H), 2.52 (br. s., 4H), 1.66-1.83 (m, 6H), 1.49 (d, J=7.53 Hz, 2H), 0.98(t, J=7.40 Hz, 3H).

Formula I2-butoxy-7-(4-(pyrrolidin-1-ylmethyl)benzyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine

To 20 L clave were added the compound of formula VIII (440.0 g, 1.11mol) and dichloromethane (7.0 L), and the temperature of the system waslowered to below −15° C. with stirring. After triethylsilane (880 mL,5.55 mol) was added dropwise, trifluoroacetic acid (880 mL) was addeddropwise, with the temperature kept below −10° C. during addition. Afteraddition, the reaction was carried out at 0° C. for 2 h and monitored byliquid chromatography until the raw material point disappeared. Aftercompletion of the reaction, the reaction liquid was concentrated todryness, and 2.2 L of ethyl acetate was added to the solution. Stirringwas performed to lower the temperature to below 0° C. Then saturatedsodium carbonate solution was added to adjust the solution to pH 9-10,during which the system temperature was kept below 10° C. Filtration wasperformed and the resulting filter cake was slurried with 2.2 L ofwater. Filtration was performed and drying was conducted under reducedpressure to give 550 g of trifluoroacetate of the compound of formula Ias white solid.

To 1.6 L of ethanol was added 525 g of trifluoroacetate of the compoundof formula I as white solid, and the temperature of the system waslowered to about 0° C. with stirring. Then 2.2 L of 1 mol/L sodiumhydroxide solution was added. Filtration was performed and the resultingfilter cake was slurried with 2.5 L of purified water. Filtration wasperformed and drying was conducted under reduced pressure to give 380.0g of the compound of formula I as solid.

¹H NMR (400 MHz, methanol-d₄) δ 7.27 (d, J=8.0 Hz, 2H), 7.22 (d, J=8.0Hz, 2H), 7.04 (s, 1H), 4.32 (t, J=6.6 Hz, 2H), 3.99 (s, 2H), 3.60 (s,2H), 2.55-2.52 (m, 4H), 1.85-1.71 (m, 6H), 1.55-1.48 (m, 2H), 1.00 (t,J=7.4 Hz, 3H).

Example 1: Preparation of Crystal Form A of2-butoxy-7-(4-(pyrrolidin-1-ylmethyl)benzyl)-5H-pyrrolo[3,2-d]pyrimidin-4-amine

To ethanol (2.3 L) was added the compound of formula I (380 g) obtainedin preparation example 1, and the mixture was heated reflux and stirredto be dissolved completely. After filtration, the filtrate was cooled toroom temperature and allowed to stand for crystallization. Filtrationwas performed, and the obtained filter cake was dried under reducedpressure to give 310.0 g solid, i.e. crystal form A of the compound offormula I.

XRPD was measured as follows: apparatus: Bruker D8 ADVANCE X-raydiffractometer; method: target: Cu: K-Alpha; wavelength λ=1.54179 Å;voltage: 40 kV; current: 40 mA; scanning range: 4˜40°; sample rotationspeed: 15 rpm; scanning speed: 10°/min.

The obtained compound crystal had diffraction peaks substantially asshown in FIG. 1.

Example 2: High Temperature Stability Test

The crystal form A of the compound of formula I was tested in theaccelerated test under high temperature conditions for stability inaccordance with Guidelines for the Stability Test of PharmaceuticalIngredients and Pharmaceutical Preparations (Chinese Pharmacopoeia 2010Appendix XIXC).

The crystal form A prepared in Example 1 was placed in an open-cleancontainer at 60° C. The samples were taken for test on day 10, day 20and day 30, respectively. The results were compared with the initialtest result on day 0, and the results were shown in Table 1.

TABLE 1 Total content Sampling time Content of impurities (day)Appearance (%) (%) 0 White powder 99.8 0.92 10 Light yellow powder 98.51.10 20 Light yellow powder 98.1 1.18 30 Light yellow powder 98.8 1.29

It was shown in high temperature stability test that, the crystal form Aof the compound of formula I has good stability in high temperatureconditions.

Example 3: High Humidity Stability Test

The crystal form A of the compound of formula I was tested in theaccelerated test under high humidity conditions for stability inaccordance with Guidelines for the Stability Test of PharmaceuticalIngredients and Pharmaceutical Preparations (Chinese Pharmacopoeia 2010Appendix XIXC).

The crystal form A prepared in Example 1 was subjected to an acceleratedtest in a constant temperature and humidity vessel under the conditionsof 40° C./75% humidity (open). The samples were taken for test on day30, day 60 and day 90, respectively. The results were compared with theinitial test results on day 0, and the results were shown in thefollowing Table 2.

TABLE 2 Total content Test Sampling time Content of impuritiesconditions (day) Appearance (%) (%) 40° C./75% 0 White powder 100.2 0.92humidity(open) 30 White powder 99.9 0.92 60 White powder 99.9 0.65 90White powder 100.2 0.93

It was shown in high humidity stability test that, the crystal form A ofthe compound of formula I has good stability in high humidityconditions.

Examples of Pharmaceutical Activity

Efficacy example 1: Toll-like receptor 7 and Toll-like receptor 8 invitro receptor binding activity screen

Reagents:

HEK-blue hTLR7 cell and HEK-blue hTLR8 cell (available from InvivoGen)

DMEM medium

heat inactivated fetal bovine serum

Anti Mycoplasma reagent Normocin™

bleomycin

blasticidin

The structure of GS-9620 and R848 used are as follows, wherein thepreparation of GS-9620 could be referred to the process disclosed in0520100143301; R848 was commercially available from ABGENT (IMG-2208,specification: 0.5 mg).

Scheme:

1. Preparation of 96-well compound plate:

The compounds were gradient diluted with DMSO in 3-fold using liquidwork station POD starting at a concentration of 10 mmol/L and 10 pointswere diluted (2nd column to 11th column, and each point was duplicated).At 12th column, 1 μL of 5 mg/mL positive compound R848 was added aspositive control; and at 1st column, 1 μL of DMSO was added as negativecontrol. Each well contained 1 μL of DMSO.

2. The cells in culture flask were collected and the cell density wasdiluted to 250,000 cells/mL.

3. 200 μL (50,000 cells/well) of cell suspension was added into preparedcompound plate and the final concentration of DMSO in each well was0.5%.

4. The culture plates containing cells and the compounds were incubatedin CO₂ incubator for 24 h at 37° C., 5% CO₂.

5. After 24 h incubation, 20 μL of supernatant was removed from eachwell to a 96-well transparent assay plate. To each well of the assayplate was added 180 μL of Quanti-Blue reagent and the plate wasincubated in an incubator at 37° C., 5% CO₂ for 1 h.

6. After 1 h, the content of alkaline phosphatase in 20 μL ofsupernatant was determined using Microplate Reader OD650.

7. EC₅₀ of each compound was obtained with Prism software.

Results were shown in Table 3.

TABLE 3 Name of the TLR7 EC50 TLR8 EC50 compound (nM) (nM) GS-9620 5177867 Compound of 160 11632 formula I

The compound of formula I according to the invention showed higher invitro receptor binding activity to Toll-like receptor 7 than the control(Toll-like receptor 7 agonist GS-9620) and lower in vitro receptorbinding activity to Toll-like receptor 8 than the control (Toll-likereceptor 7 agonist GS-9620). The compound of the present invention hasdistinct selectivity differences with respect to different receptors,and the effect is superior over the prior art.

Efficacy Example 2: Peripheral Blood Mononuclear Cell Test Scheme

The purpose of this example is to determine the expression level ofcytokines 24 h after stimulation to human peripheral blood mononuclearcells (PBMC) with the compound of formula I.

The cell supernatant was assayed without dilution and the levels ofIFN-α were directly determined. The compound of formula I was firstlyformulated into 20 mM DMSO stock solution during the test and wasgradient diluted with cell medium in 10-fold with the total number of 11diluting points. The compounds in 9 diluting points (the highestconcentration was 200 μmol/L) were added into 96-well plate with 50 μLin each well. Fresh human peripheral blood mononuclear cells wereinoculated, with 150 μL in each well containing 450,000 cells. The cellculture plate was incubated in an incubator at 37° C., 5% CO₂ for 24 h.After incubation, the culture plate was centrifuged at 1200 rpm for 5min and the supernatant was collected and stored at −20° C. fordetermination. The determination of cytokine was performed usingCytometric Bead Array (CBA) of BD-Pharmingen on flow cytometer. Usingthe above determining method, the lowest drug concentration stimulatingthe production of 30 pg/mL of IFN-α was designated as the MEC value inthe cytokine stimulating test. The results were shown in Table 4.

TABLE 4 Name of the IFN-α MEC TNF-α MEC compound (nM) (nM) GS-9620 50500 Compound of 5 500 formula I

Compared with the control (GS-9620), the compound of formula I of theinvention showed better in vitro IFN-α inducing activity of PBMCs andcomparable TNF-α inducing activity.

SEQUENCE LISTING

Not Applicable

The invention claimed is:
 1. A crystal form A of a compound of formulaI:

wherein the crystal form A is characterized by an X-ray powderdiffraction pattern having diffraction peaks (2θ) at 5.5°±0.2°,10.1°±0.2°, 13.8°±0.2°, 16.4°±0.2°, 19.7°±0.2°, 23.7°±0.2°, 24.1°±0.2°,and 27.9°±0.2°.
 2. The crystal form A according to claim 1, wherein thecrystal form A is further characterized by an X-ray powder diffractionpattern having diffraction peaks (2θ) at 5.5°±0.2°, 10.1°±0.2°,13.8°±0.2°, 16.4°±0.2°, 17.9°±0.2°, 19.0°±0.2°, 19.7°±0.2°, 20.3°±0.2°,21.8°±0.2°, 22.1°±0.2°, 23.7°±0.2°, 24.1°±0.2°, 25.5°±0.2°, 27.9°±0.2°,32.9°±0.2°, and 34.0°±0.2°.
 3. The crystal form A according to claim 1,wherein the crystal form A is further characterized by an X-ray powderdiffraction pattern as shown in FIG.
 1. 4. The crystal form A accordingto claim 1, wherein the crystal form A is further characterized by adifferential scanning calorimetry thermogram having an initialtemperature of 199.0° C.±5° C. and a peak temperature of 200.4° C.±5° C.5. A composition comprising a carrier or excipient and at least 95% w/wof the crystal form A according to claim 1, based on a total weight ofthe composition.
 6. A composition comprising a carrier or excipient andat least 50% w/w of the crystal form A according to claim 1, based on atotal weight of the composition.
 7. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or excipient and atherapeutically effective amount of the crystal form A according toclaim
 1. 8. A method for inhibiting toll-like receptor activity in asubject, wherein the method comprises administering to the subject inneed thereof a therapeutically effective amount of the crystal form Aaccording to claim
 1. 9. The method according to claim 8, wherein thesubject has a viral infection.
 10. The method according to claim 8,wherein the subject has hepatitis B or hepatitis C.
 11. A method forinhibiting toll-like receptor activity in a subject, wherein the methodcomprises administering to the subject in need thereof a therapeuticallyeffective amount of the pharmaceutical composition according to claim 7.12. The method according to claim 11, wherein the subject has a viralinfection.
 13. The method according to claim 11, wherein the subject hashepatitis B or hepatitis C.
 14. A process for preparing the crystal formA according to claim 1, wherein the process comprises the followingsteps: 1) dissolving the compound of formula I according to claim 1 in acrystallizing solvent to form a solution, wherein the crystallizingsolvent is selected from the group consisting of methanol, ethanol,n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, acetone,ethyl acetate, and water, or a co-solvent mixture of any two or morethereof; 2) heating the solution of step 1) to reflux while stirring; 3)cooling the solution of step 2) to room temperature; 4) filtering thesolution of step 3) to obtain a filter cake; and 5) drying the filtercake of step 4) to obtain the crystal form A according to claim
 1. 15.The process according to claim 14, wherein the crystallizing solvent instep 1) is ethanol.
 16. The process according to claim 14, wherein instep 1), the amount of crystallizing solvent added per gram of thecompound of formula I is 2 mL to 10 mL.
 17. The process according toclaim 14, wherein in step 1), the amount of crystallizing solvent addedper gram of the compound of formula I is 4 mL to 8 mL.
 18. The processaccording to claim 14, wherein in step 1), the amount of crystallizingsolvent added per gram of the compound of formula I is 5 mL to 7 mL. 19.A crystal form A of a compound of formula I:

wherein the crystal form A is characterized by an X-ray powderdiffraction pattern having diffraction peaks (2θ) at 5.5°±0.2°,10.1°±0.2°, 13.8°±0.2°, 16.4°±0.2°, 19.7°±0.2°, 23.7°±0.2°, 24.1°±0.2°,and 27.9°±0.2°; and further wherein the crystal form A has the followingcell parameters: a=16.560 (3) Å; b=10.426 (2) Å; c=12.203 (2) Å; α=90°;β=98.54(3)°; γ=90°; Space group P2₁/c; and Z=4.
 20. The crystal form Aaccording to claim 19, wherein the crystal form A is furthercharacterized by an X-ray powder diffraction pattern having diffractionpeaks (2θ) at 5.5°±0.2°, 10.1°±0.2°, 13.8°±0.2°, 16.4°±0.2°, 17.9°±0.2°,19.0°±0.2°, 19.7°±0.2°, 20.3°±0.2°, 21.8°±0.2°, 22.1°±0.2°, 23.7°±0.2°,24.1°±0.2°, 25.5°±0.2°, 27.9°±0.2°, 32.9°±0.2°, and 34.0°±0.2°.